Cannabinoid Combination TargetsNOTCH1-Mutated T-ALL Through the Integrated Stress Response Pathway

In T-cell acute lymphoblastic leukemia (T-ALL) more than 50% of cases display autoactivation of Notch1 signaling, leading to oncogenic transformation. We have previously identified a specific chemovar of Cannabis that induces apoptosis by preventing Notch1 maturation in leukemia cells. Here, we isolated three cannabinoids from this chemovar that synergistically mimic the effects of the whole extract. Two were previously known, Cannabidiol (CBD) and Cannabidivarin (CBDV); whereas the third cannabinoid, which we termed 331-18A, was identified and fully characterized in this study. We demonstrated that these cannabinoids act through Cannabinoid receptor type 2 and TRPV1 to activate the integrated stress response pathway by depleting intracellular Ca²⁺. This is followed by increased mRNA and protein expression of ATF4, CHOP and CHAC1, which is hindered by inhibiting the upstream initiation factor eIF2α. The increased abundance of CHAC1 prevents Notch1 maturation, thereby reducing the levels of the active Notch1 intracellular domain, and consequently decreasing cell viability and increasing apoptosis. Treatment with the three isolated molecules resulted in reduced tumor size and weight in-vivo and slowed leukemia progression in mice models. Altogether, this study elucidated the mechanism of action of three distinct cannabinoids in modulating the Notch1 pathway, and constitutes an important step in the establishment of a new therapy for treatingNOTCH1-mutated diseases and cancers such as T-ALL.